Surface polysaccharides of pathogenic bacteria, including capsular polysaccharides (CPS) or the O-specific polysaccharide (O-SP) of lipopolysaccharides (LPS), serve both as essential virulence factors and as protective antigens. Covalent binding of these saccharides to medically-useful proteins to form conjugates both increases their immunogenicity and confers to them T-cell dependence making them suitable vaccines for infants and children. The O-SP of Shigella sonnei bound to recombinant non toxic P. aeruginosa exoprotein A (rEPA) had an efficacy of over 70% in young adults exposed to 6-14% attack rates. This conjugate and of S. flexneri 2a bound to the succinylated exoprotein A (rEPA-succ) were safe and induced IgG antibodies to the homologous LPS in 1-4 year-olds. A randomized, blinded, phase 3 study of these conjugates in 1-4 year-olds with each conjugate serving as a control for the other showed these vaccines to be safe but did not show efficacy. Fold increases in antibody levels were, as before, similar to those of adults but the actual levels achieved were lower.[unreadable] [unreadable] The effect of these childrens immune sera and IgG isolated from them upon Shigella invasion into epithelial intestinal cells was studied in vitro using Caco-2 and HeLa cells. The sera inhibited Shigella invasion in a type specific manner. Pretreatment of the sera or of Caco-2 cells with O-SP abrogated these effects in a type specific and dose dependent manner.[unreadable] [unreadable] To enhance the immunogenicity of these conjugates S. sonnei and S. flexneri 2a O-SPs were bound to additional carrier proteins: tetanus toxoid (TT) and recombinant protective antigen (rPA). Mice were injected twice, with the same or a different carrier the second time. For S. flexneri 2a the TT conjugate injected twice induced the highest (>4-fold) antibody levels. For S. sonnei, the TT conjugate followed by the rPA conjugate induced significantly higher levels than the other combinations. This effect is investigated further.[unreadable] [unreadable] Modification of O-SP by glycosylation or acetylation is important for its antigenic specificity and immunogenicity. The degree of O-acetylation and localization of the O-acetyl groups and glucose substitution of S. flexneri 2a O-SP were investigated. On the basis of these studies, the structure of the repeat unit was assigned.